Project Summary: While previously unrecognized, it is now documented that having delivered a child within 10 years is an independent predictor of early onset breast cancer and poor outcomes. These cancers, based largely on work from our labs, is now referred to as postpartum breast cancer (PPBC), and represents a significant clinical problem. Every year in the US alone, ~15,000 young women (accounting for ~50% of all young women?s breast cancer cases) are diagnosed during the high risk, postpartum period. Research in our lab has advanced the hypothesis that weaning-induced mammary gland involution is the driver of increased incidence and metastasis in PPBC patients. During the transitory window of weaning-induced involution, the normal involuting mammary gland is skewed towards a pro-tumor microenvironment, as it exhibits increased lymphangiogenesis, fibroblast activation, and extracellular matrix deposition; as well as enrichment for myeloid derived suppressor cells (MDSC), M2-skewed macrophages, and Th-17, Th-2 and Treg skewed T cells compared to nulliparous glands. These discoveries support our hypothesis that sub-clinical, non-life threatening disease progresses to overtly invasive cancer during weaning-induced involution. In multiple murine models, we confirm that the tissue microenvironment of the involuting gland increases mammary tumor incidence and progression. Further, we demonstrate that ibuprofen (IBU) treatment targeted to the involution window blocks >50% of these tumors from emerging, depending on model. Further, of those tumors that do emerge, progression to metastatic disease is suppressed. One mechanism by which IBU appears to inhibit PPBC is through anti-tumor immunity, as IBU increases the number of cytotoxic T cells, consistent with increased immune surveillance. Importantly, we find the ability of IBU to restore the adaptive arm of the immune system occurs in the absence of adverse host autoimmune reactions and does not impact the ability of dams to successfully nurse their young in subsequent pregnancies. These findings support clinical utility of IBU in the setting of a PPBC prevention trial. Our studies also identify opportunities to improve efficacy, and vitamin D (Vit D) is proposed based on its anti-tumor and anti-inflammatory activity, and its safety and particular relevance in postpartum women, who are at high risk for VitD deficiency. Here we propose to 1) investigate the efficacy of IBU treatment in combination with vitamin D, in rodent models of PPBC with improved human relevance, 2) identify the mechanism by which IBU +/- VitD mitigates the pro-tumorigenic microenvironment of the involuting gland with the goal of identifying additional involution-specific targets; and 3) translate our rodent studies defining the ?immune composition of the ?at risk? involuting mammary gland to women, by defining the immune milieu in the normal human breast across reproductive states. Only through understanding how the normal, involuting human breast confers increased BrCa risk, can we identify additional targets to improve the efficacy of our chemoprevention strategy and lay the foundation for PPBC prevention.